SUMMARY OF WORK: The goal of this project is to investigate molecular alterations in cell cycle control during neoplastic transformation. In Subproject 1, cell cycle control in v-mos-transformed NIH3T3 cells is being investigated. These cells are unable to exit the proliferative cycle into quiescence following serum removal. Instead they arrest in early G1 and express abnormally highlevels and/or have abnormal kinase activity associated with p34cdc2, p33cdk2, cyclin D1, cyclin E, and cyclin A. Inducible mos expression in serum starved NIH3T3 cells resulted in increased expression of p34cdc2 that did not correlate with MAP kinase activation but did correlate with the activation of S-phase E2F complexes. In Subproject 2, the molecular mechanism of the G2 cell cycle checkpoint response to ionizing radiation (IR) is being analyzed in normal human fibroblasts and in fibroblasts that lack normal function of p53, pRB, or the ataxia telangiectasia (AT) cancer susceptibility gene product. Normal human fibroblasts respond to exposure to IR by rapidly delaying entry into mitosis with an associated strong inhibition of p34cdc2/cyclinB protein kinase activity. AT fibroblasts exposed to IR show little delay of entry into mitosis or inhibition of kinase activity. The rapid G2 checkpoint response to IR does not require p53, pRB, or p21 function. However, lack of p53 results in a progressively increasing proportion of cells losing G2 checkpoint function correlated with an increasing proportion of cells with chromosomal abnormalities. We are particularly interested in the role of the ATM gene product in the G2 checkpoint and how it signals from broken DNA to the inactivation of p34CDC2/cyclinB protein kinase complexes. We have developed and characterized several antisera to the ATM protein and are investigating its expression and function. In addition to aiding our understanding of the process of carcinogenesis, these studies hold great potential for providing insight into the mechanism of action of environmental toxins, especially non-genotoxic carcinogens.